Gilchrist Erin J, O'Neil Nigel J, Rose Ann M, Zetka Monique C, Haughn George W
Department of Botany, 6270 University Blvd, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
BMC Genomics. 2006 Oct 18;7:262. doi: 10.1186/1471-2164-7-262.
TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetic technique based on the use of a mismatch-specific enzyme that identifies mutations in a target gene through heteroduplex analysis. We tested this technique in Caenorhabditis elegans, a model organism in which genomics tools have been well developed, but limitations in reverse genetics have restricted the number of heritable mutations that have been identified.
To determine whether TILLING represents an effective reverse genetic strategy for C. elegans we generated an EMS-mutagenised population of approximately 1500 individuals and screened for mutations in 10 genes. A total of 71 mutations were identified by TILLING, providing multiple mutant alleles for every gene tested. Some of the mutations identified are predicted to be silent, either because they are in non-coding DNA or because they affect the third bp of a codon which does not change the amino acid encoded by that codon. However, 59% of the mutations identified are missense alleles resulting in a change in one of the amino acids in the protein product of the gene, and 3% are putative null alleles which are predicted to eliminate gene function. We compared the types of mutation identified by TILLING with those previously reported from forward EMS screens and found that 96% of TILLING mutations were G/C-to-A/T transitions, a rate significantly higher than that found in forward genetic screens where transversions and deletions were also observed. The mutation rate we achieved was 1/293 kb, which is comparable to the mutation rate observed for TILLING in other organisms.
We conclude that TILLING is an effective and cost-efficient reverse genetics tool in C. elegans. It complements other reverse genetic techniques in this organism, can provide an allelic series of mutations for any locus and does not appear to have any bias in terms of gene size or location. For eight of the 10 target genes screened, TILLING has provided the first genetically heritable mutations which can be used to study their functions in vivo.
定向诱导基因组局部突变（TILLING）是一种反向遗传学技术，该技术基于使用错配特异性酶，通过异源双链分析来识别目标基因中的突变。我们在秀丽隐杆线虫中测试了这项技术，秀丽隐杆线虫是一种模式生物，其基因组学工具已得到充分发展，但反向遗传学的局限性限制了已鉴定的可遗传突变的数量。
为了确定TILLING是否代表一种对秀丽隐杆线虫有效的反向遗传学策略，我们构建了一个约1500个个体的经甲基磺酸乙酯（EMS）诱变的群体，并筛选了10个基因中的突变。通过TILLING共鉴定出71个突变，为每个测试基因提供了多个突变等位基因。所鉴定的一些突变预计为沉默突变，这要么是因为它们位于非编码DNA中，要么是因为它们影响密码子的第三个碱基而不改变该密码子编码的氨基酸。然而，所鉴定的突变中有59%是错义等位基因，导致基因蛋白质产物中的一个氨基酸发生变化，3%是推定的无效等位基因，预计会消除基因功能。我们将通过TILLING鉴定的突变类型与先前正向EMS筛选报道的突变类型进行了比较，发现96%的TILLING突变是G/C到A/T的转换，这一比率显著高于正向遗传筛选中观察到的比率，在正向遗传筛选中还观察到了颠换和缺失。我们获得的突变率为1/293 kb，这与在其他生物体中观察到的TILLING突变率相当。
我们得出结论，TILLING是秀丽隐杆线虫中一种有效且经济高效的反向遗传学工具。它补充了该生物体中的其他反向遗传学技术，可以为任何基因座提供一系列突变等位基因，并且在基因大小或位置方面似乎没有任何偏向性。对于所筛选的10个目标基因中的8个，TILLING提供了首个可遗传的突变，可用于在体内研究它们的功能。
